Abstract

Calcitonin is a 32-amino acid peptide, which causes a decrease in blood calcium levels and bone resorption in the human body. It is produced in different species. Salmon calcitonin is used as a medicine for diseases such as osteoporosis, Paget’s disease, and hypercalcemia. However, the salmon calcitonin peptide used as a medicine, could induce immune responses in humans. Decreasing the antigenicity of salmon calcitonin could improve this molecule for pharmaceutical usage. In this study, improving physicochemical properties and reducing allergenicity and especially the antigenicity of salmon calcitonin were followed. The calcitonin sequences of different species were evaluated, and those with better properties were considered as a guide for peptide engineering. In silico methods were utilized to characterize the properties of the reviewed calcitonin sequences, and the best sequences (the calcitonin of sheep, dog, rat, and human) were used as a template to decrease the antigenicity of salmon calcitonin. The epitopic parts, i.e., amino acids 16 to 29, were identified by different servers. Hot spot residues including Y22, N26, T27, and S29 were characterized based on the main criteria of being a B-cell epitope including convexity index, hydrophilicity, and surface accessibility. These residues were replaced with lower antigenic counterparts. Results showed the final selected mutant (T27V/S29V) had a lower antigenicity and higher solubility and stability than salmon calcitonin. Thus, our suggested mutant could be a potential alternative candidate to salmon calcitonin. However, future in vitro and in vivo evaluations are needed to confirm its suitability for clinical usage.

中文翻译：

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引入改进的降钙素突变体的肽工程方法

摘要

降钙素是一种由 32 个氨基酸组成的肽，会导致血钙水平降低和人体内骨吸收。它产生于不同的物种。鲑鱼降钙素可用作治疗骨质疏松症、佩吉特氏病和高钙血症等疾病的药物。然而，用作药物的鲑鱼降钙素肽可以诱导人类的免疫反应。降低鲑鱼降钙素的抗原性可以改善该分子的药物用途。在本研究中，改善鲑鱼降钙素的理化性质并降低过敏性，特别是抗原性。对不同物种的降钙素序列进行了评估，将具有更好特性的降钙素序列视为肽工程的指南。利用计算机方法来表征所审查的降钙素序列的特性，并使用最佳序列（绵羊、狗、大鼠和人的降钙素）作为模板来降低鲑鱼降钙素的抗原性。表位部分，即氨基酸16至29，由不同的服务器鉴定。根据 B 细胞表位的主要标准（包括凸度指数、亲水性和表面可及性）对包括 Y22、N26、T27 和 S29 在内的热点残基进行了表征。这些残基被较低抗原性的对应物取代。结果表明，最终选择的突变体（T27V/S29V）比鲑鱼降钙素具有更低的抗原性和更高的溶解度和稳定性。因此，我们建议的突变体可能是鲑鱼降钙素的潜在替代候选者。然而，需要未来的体外和体内评估来确认其是否适合临床使用。